A new class of nucleic acid labeling reagents has been developed that facilitates the covalent attachment of a variety of non-isotopic labels in a simple one-step reaction. Using these reagents both fluorescent (fluorescein, rhodamine, Cy3, and Cy5) and non-fluorescent (biotin, digoxin, dinitrophenol) detectable labels can be attached to DNA or RNA sequences in a non-destructive manner. In the Phase I research, a series of experiments were performed in which the labeling reagents were: 1) optimized for efficient DNA and RNA labeling; 2) tested for stability under both storage conditions and hybridization conditions; and 3) optimized for their ability to be used in in vitro hybridization reactions (i.e., slot blots, southern blots, and northern blots). In the Phase II research, these non-radioactive labeling reagents will be tested and optimized for use in fluorescence in situ hybridization (FISH) applications. The applicants state that their utility as efficient, easy to use nucleic acid labeling reagents is clear. They also state that their use in labeling will facilitate a systematic approach to probe formation, that will for the first time, help to delineate the critical attributes needed to obtain the highest efficient in situ hybridization probes (i.e., optimal probe length, optimal probe length to labeling ratio). PROPOSED COMMERCIAL APPLICATION: NOT AVAILABLE